The zinc finger protein family encompasses a broad variety of proteins with assorted functions. One relatively uncommon class of zinc finger proteins contains fingers of the Cys-Cys-Cys-His (SEQ ID NO:48) (CCCH) type, in which three cysteines and one histidine are thought to coordinate a single atom of zinc. Members of a very small subclass of the larger family of CCCH zinc finger proteins contain two tandem zinc fingers consisting of Cx8Cx5Cx3H (SEQ ID NO:47) (wherein “x” refers to variable amino acids), spaced exactly 18 amino acids apart. The prototype of proteins of this CCCH double zinc finger subclass is tristetraprolin (TTP), also known as TIS11 and Nup475. TTP is localized to the nucleus of quiescent fibroblasts, but is rapidly phosphorylated on serine residues and translocated to the cytosol after stimulation with serum or other mitogens. TTP is almost completely cytosolic in macrophages.
TTP-deficient mice appear normal at birth, but rapidly develop a wasting syndrome accompanied by erosive arthritis, dermatitis, alopecia, autoantibodies and myeloid hyperplasia. Essentially all of these inflammation-associated conditions can be prevented by the injection of monoclonal antibodies specific for mouse tumor necrosis factor-α (TNF-α), one of the principal mediators of the inflammatory response in mammals.
In addition to its well known role in acute septic shock, TNF-α has been implicated in the pathogenesis of chronic processes such as autoimmunity, graft-versus-host disease, rheumatoid arthritis, Crohn's disease, and the cachexia that accompanies cancer and acquired immunodeficiency syndrome (AIDS). Anti-TNF-α therapies such as neutralizing antibodies against TNF-α and chimeric soluble TNF-α receptors have demonstrated efficacy against some of these conditions in clinical trials.
The present invention is based upon the discovery that the TTP tandem zinc finger domain alone (as well as the analogous domain from other TTP-related polypeptides) is sufficient to bind to an AU-rich element (ARE) within the 3′ untranslated region (UTR) of a TNF-α mRNA molecule, thereby targeting the TNF-α mRNA molecule for destruction. The present invention is further based upon the discovery that this mRNA turnover mechanism is not unique to TNF-α mRNA, as destruction of other mRNAs containing AREs, such as the mRNA that encodes granulocyte-macrophage stimulating factor (GM-CSF), is stimulated by the tandem zinc finger domain of TTP and TTP-related polypeptides. Accordingly, the invention provides functional fragments of TTP and TTP-related polypeptides, nucleic acids encoding such functional fragments, and methods of using the nucleic acids and polypeptide fragments to regulate (i.e., stimulate or inhibit) the destruction of mRNAs that contain an ARE. These methods may be used to treat diseases and conditions that are affected by polypeptides encoded by an mRNA molecule whose destruction is modulated by an ARE. The polypeptides and methods of the invention can also be used to identify compounds that regulate the activity of TTP and TTP-related polypeptides, and are therefore useful for regulating levels of mRNA encoding TNF-α, GM-CSF, and other mRNAs containing functional AREs, such as that encoding interleukin-3 (IL-3).